|
BABB (Becker et al, 2008; Dent et al, 1989; Dodt et al, 2007; Spalteholz, 1914)
|
Scale (Hama et al, 2011)
|
Tetrahydrofuran (THF) (Ertürk et al, 2012b) |
3DISCO (Becker et al, 2012; Ertürk et al, 2012a) |
CLARITY (Chung et al, 2013; Lee et al, 2014; Tomer et al, 2014)
|
PACT (passive CLARITY technique ) / PARS (perfusion-assisted agent release in situ) (Yang et al, 2014)
|
|
| Fixation method and other tissue preparation |
PFA perfusion |
PFA perfusion |
PFA perfusion |
PFA perfusion |
PFA, acrylamide, bis-acrylamide, VA044 perfusion |
PFA perfusion and subsequent hydrogel / initiator perfusion |
| Dehydration |
Yes |
No |
Yes |
Yes |
No |
No |
| Clearing solution |
Benzylalcohol, benzylbenzoate |
ScaleA2: Urea, glycerol, Triton X-100 |
THF, dichloromethane (DCM), benzylalcohol, benzylbenzoate |
THF, DCM, dibenzyl ether (DBE) |
Active transport organ-electrophoresis approach -electrophoretic tissue clearing (ETC) in sodium borate buffer containing 4% SDS |
Up to 2 weeks 8%SDS in PBS, pH 7.5 at 37°-42℃ followed by extensive PBS perfusion washing over 2-3 days |
| Time of protocol |
Up to 3 days or more |
3 days - several weeks (- months) |
∼1 day |
∼1 day |
∼7-21 days |
2-3 days for most organs, 1-2 weeks for whole brain |
| Tissue clearing principle (RI matching) |
Dehydration |
Increased RI of aqueous phase |
Dehydration and lipid removal (THF) |
Dehydration and lipid removal (DCM) |
Ionic lipid extraction (Passive or electrophoretic) |
Ionic lipid extraction |
| Fluorescence quenching |
High (Dehydration) |
Minimal |
High (Dehydration) |
Continuous quenching in final clearing solution |
No |
No |
| Main Disadvantages |
Not compatible with tissues containing a high degree of lipids, fluorescence quenching, autofluorescence, benzylbenzoate and benzylalcohol are toxic and dissolve plastic |
Long clearing process, fragility of cleared samples, sample expansion |
Impaired ultrastructure, loss of cellular and molecular information due to lipid removal, benzylbenzoate and benzylalcohol are toxic and dissolve plastic |
Quenching in final clearing solution requires immediate imaging, loss of cellular and molecular information due to lipid removal |
Passive: Long clearing Electrophoretic: Additional equipment needed (platinum electrodes), possible epitope loss, heating |
Relatively slow for large organs like whole brain (compared to BABB, Scale, THF or 3DISCO), perfusion chamber required |
| Main Advantages |
Accessibility to reagents |
Signal preservation of fluorescent proteins, adjustable formula for various organs, low toxicity of chemicals |
Reduced fluorescence quenching, antibody use possible, but long incubation times required |
Relatively low fluorescence quenching, lipid removal, antibody use possible, but long incubation times required |
Preservation of biological information by providing physical framework, no quenching due to ionic extraction technique, deep antibody penetration with long incubation times |
Superior to CLARITY due to lack of electrophoresis and reduction of tissue degradation, more cost effective than CLARITY, reduced tissue swelling, fast simultaneous clearing of multiple tissues |
